Thermolysin was first found in the culture broth of Bacillus thermoproteolyticus (Endo, S. (1962) J. Fermentation Tech., 40, 346-353) and a number of investigations have been conducted thereon. Thus, for instance, its amino acid sequence (Titani, K., et al., (1972) Nature New Biol., 238, 35-37) and the three-dimensional structure of the enzyme (Holmes, M. A. and Matthews, B. W., (1982) J. Mol. Biol. 160., 623-639) have been elucidated. Meanwhile, the protease gene was cloned from Bacillus thermoproteolyticus (EP-A-0418625) and the amino acid sequence of the mature enzyme as deduced from the nucleotide sequence of said gene was found to be different from the original primary structure as indicated by Titani in two positions. Thus, it was reported that the 37th (from the amino terminal) amino acid residue of the mature enzyme is not aspartic acid but asparagine and the 119th one is not glutamic acid but glutamine. This amino acid sequence is identical with that coded by nprM, one of the protease genes cloned from Bacillus stearothermophilus (Kubo, M., et. al., (1988) Journal of General Microbiology 134, 1883-1892).
Therefore, in the present specification, the protease coded by this nprM gene or the gene from Bacillus thermoproteolyticus is referred to as "wild type thermolysin-like neutral metallo-protease".
Alteration of specific activity and stability of thermolysin-like neutral metallo-protease has very recently been reported (Kubo M., et al., (1992) Applied and Environmental Microbiology, 58, 3779-3783). In this article various mutants have been described which differ in one or more amino acid residues in the primary structure, especially at positions 93, 110, 114, 115, 136, 137, 143, 151, 157, 193, 211, 217 and 221. But in this reference, the activity was measured only by casein digestion method. None of these mutants, however, did show any substantially improved activity in relation to Z-APM synthesis or digestion. It now (as described further in the examples below) also has been established that the activity for casein digestion does not correlate to that for Z-APM synthesis: it appears that even if the specific activity for casein digestion increases, the specific activity for Z-APM synthesis does not always increase.
Based on these observations, and because there are various problems in the enzymatic synthesis of Z-APM, such as relatively low activity of the enzyme, inactivation of the enzyme during the condensation reaction and hydrolysis of the product Z-APM and the starting material L- or D,L-phenylalanine methyl ester (PM), due to the long reaction time, and/or unfavorable pH conditions, there is need to develop improved enzymes that have higher activity for the synthesis of Z-APM than wild-type thermolysin. Of course where PM is mentioned in this application also its salts are included in the meaning of the term PM.